Project Summary Multiple myeloma is a uniformly fatal plasma cell cancer that kills over 11,000 individuals annually in the U.S. alone. No cure exists, and improved therapies are desperately needed. We have recently discovered that chaetocin, a natural product previously unreported to have anti- cancer effects, has potent and selective in vitro, ex vivo and in vivo anti-myeloma activity. Impressively, the anti-myeloma effects of chaetocin are maintained in dexamethasone- or doxorubicin-resistant myeloma cells and in patient myeloma samples collected from heavily pre-treated patients harboring a diverse array of cytogenetic alterations. Mechanistically, chaetocin is rapidly and dramatically accumulated in cancer cells, requiring intact/unreduced disulfides for uptake, and imposition of ROS for cytotoxic selectivity. Moreover, we have recently established that chaetocin is a competitive substrate for (and inhibitor of) the ROS mitigation enzyme thioredoxin reductase (TrxR) at achieved intracellular concentrations - leading to our hypothesis that TrxR may represent a cytotoxic molecular target of chaetocin. Considered collectively, chaetocin appears to represent a promising agent for further development as a potential anti-myeloma therapeutic. Based upon encouraging preliminary results, we now propose the following specific aims: Aim 1: Further evaluation of the mechanisms of chaetocin-induced cytotoxicity, cellular uptake and accumulation. We will critically evaluate the contributions of effects on TrxR and other reductases, histone modifying enzymes (including HDACs and HMTs), HIF1 and other targets to the cytotoxic selectivity of chaetocin; as well as further investigate mechanisms of cellular chaetocin uptake and accumulation. Aim 2: Further clarification of the cytotoxic selectivity of chaetocin with systematic exploration of differential functions of ROS pathways in myeloma cells. We will further define the selectivity of chaetocin in normal vs. malignant hematological cells and, in close coordination with mechanistic studies in Aim1, probe the molecular basis of observed cytotoxic selectivity through elaboration of differential activities of ROS and other pathways in paired chaetocin sensitive/resistant patient cells and myeloma cell lines. Aim 3: Further evaluation of the in vivo toxicities and efficacy of chaetocin. We will optimize in vivo formulations and conduct toxicology, PK, PD, and efficacy studies in xenograft and systemic myeloma models; as well as preliminarily explore in vivo biomarkers formulated based upon mechanistic studies of Aim 1. Collectively, proposed experiments will advance understanding of the cellular effects of chaetocin, foster its further development, and provide important insights into whether other agents that similarly induce ROS may also represent attractive candidates for further development as anti-myeloma therapeutics. Project Narrative Multiple myeloma is a uniformly fatal bone marrow cancer that kills over 11,000 individuals annually in the U.S. alone; no cure exists, and improved therapies are desperately needed. We have recently discovered that chaetocin, a substance isolated from a common wood mold previously unreported to have anti-cancer effects, has potent and selective anti-myeloma activity in cancer cell lines, patient myeloma cells and in an animal model of myeloma. We now propose further studies of chaetocin intended to better define its mechanisms of action, cancer selectivity, and ability to treat myeloma in animal models in hopes of eventually developing chaetocin as a therapy for patients afflicted with myeloma.